This invention relates generally to the field of medical devices, and more particularly, to methods and compositions for sustained delivery of therapeutically-significant substances.
A number of diseases are caused by disorders in cellular metabolism. For many of these diseases the nature of the metabolic defect has been identified, and the rapid progress of biomedical research continues to further our understanding of the precise mechanisms involved. For example, Type I diabetes is known to result from defective glucose metabolism associated with decreased levels of insulin, whereas different cancers are due to defective control of cellular division and proliferation associated with mutations in a variety of cellular genes, many of which have been identified. Many disorders in cellular metabolism are caused by somatic or hereditary genetic mutations which result either in inappropriate expression of a given gene product or expression of a defective gene product. However, environmental assaults such as chemical poisoning, physical damage or biological infection can also result in specific defects in cellular metabolism. In addition, cellular aging often results in metabolic disorders. Understanding the nature of a given metabolic disorder identifies targets/goals for an effective treatment.
A traditional approach to treatment consists of administering systemically, to a patient, a pharmaceutical compound or drug that overcomes the metabolic disorder. For example, administering exogenous insulin to a patient alleviates the symptoms of Type I diabetes. There are, however, several drawbacks to this type of drug therapy. For a pharmaceutical compound to be effective, it must be administered so that it reaches its site of action at an appropriate concentration. If the compound is provided systemically, whether administered orally or by injection, undesirable side effects may be caused by the systemic levels of the compound required for it to be effective at its site of action. This is the case for many chemotherapeutic agents used to treat various forms of cancer. Current attempts to overcome this problem consist of trying to target pharmaceutical compounds to their desired site of action for sustained periods of time at effective concentrations. There is not yet a reliable and general method for such targeted drug delivery.
An additional problem with traditional drug administration is that the drug must be stable and transportable to its site of action. For many diseases, the most appropriate therapeutic compound would be a specific protein, especially if the disease results from the absence of a functional form of that protein. However, delivering any given protein to its desired site of action can be complicated by its susceptibility to denaturation and proteolytic degradation, and by poor mobility to its desired site of action.
An alternative approach is gene therapy which attempts to overcome these problems by circumventing the requirement of transporting a protein to its site of action. The goal of gene therapy is to provide DNA encoding the desired protein to the site of action. The DNA then is transcribed and translated to produce the protein in therapeutically effective concentrations at the appropriate site in the body. However, gene therapy also faces a delivery problem of how to get the DNA to the appropriate cells. Current approaches to solving this problem consist of using viral vectors, liposome encapsulation, direct injection, or complexation with carrier proteins. At present, none of these approaches provides an effective and general method for getting DNA to any desired site of action within the body. Current gene therapy technology also does not address the required duration of therapy. Hereditary diseases, for example, might require constant therapy to correct the inherited metabolic disorder. On the other hand, cancer treatment may only be needed for a time sufficient to destroy the cancer cells.
There is, therefore, a need in the art for effective devices and methods for delivering physiologically useful compounds to any desired site of action, in a controlled fashion. It is an object of the present invention to provide such devices and methods.
A novel approach to the delivery of drugs and other therapeutic substances has now been discovered. The present invention uses genetic mutations to exploit certain innate characteristics of a group of unicellular organisms known as protozoa. By genetically modifying these organisms in accordance with the present invention, the skilled practitioner can now use these organisms as improved devices for the sustained delivery of drugs and other substances.
The rationale for using these organisms as delivery devices includes the following: First, these organisms have evolved a sophisticated ability to infect a host and evade their host""s immune defenses. Consequently, these organisms can persist in their host in an undetected and undisturbed state for long periods of time. As taught herein, this characteristic can be exploited to achieve sustained, in vivo delivery of a drug or other therapeutic. Moreover, by practicing the genetic manipulations disclosed herein, an organism""s ability to persist in its host can be closely controlled. In fact, an organism can be engineered so that it can be subsequently eliminated on demand. Furthermore, the number of organisms in the host as well as the dose of drug or substance delivered to the host can also be closely controlled using the present invention.
Second, these organisms have evolved very specific, natural tissue preferences. Consequently, these organisms reside and persist in specific tissues in their respective hosts. Moreover, protozoa as a group reside in virtually every tissue or organ of vertebrates and invertebrates. As taught herein, this characteristic can be exploited to achieve targeted, tissue-specific delivery of a drug or other therapeutic substance. Currently, the only available type of targeted, sustained in vivo delivery relies on gene therapy, however, gene therapy cannot be similarly controlled, is often viral-vector mediated, and can result in undesirable and/or permanent alteration of the recipient""s genome. Thus, the teachings of the present invention overcome the limitations of gene therapy, as well as overcome numerous problems associated with more conventional drug therapy methods, such as unnecessary systemic exposure, toxicity, poor transportability, degradation and stability to name but a few.
Accordingly, the teachings of the present invention provide a device for delivery of a drug or therapeutic substance which is sustained, targeted, reversible and virus-free, without necessarily exposing the recipient to permanent genetic alterations. In one aspect, the present invention features a sustained delivery device comprising an isolated, conditionally defective unicellular organism expressing a therapeutically-significant substance. The genome of this organism is genetically altered to lack a naturally-occurring nucleotide sequence defining a genetic locus responsible for a selectable phenotype, and encode an expression product for sustained delivery. In some embodiments, the unicellular organism is a diploid organism. In other embodiments, the organism is an asexual diploid organism. In certain currently preferred embodiments, the organism is a protozoa, more preferably a parasitic protozoa. The expression product of the present device can be encoded by a heterologous gene. Currently preferred are genes encoding a hormone, enzyme or neurotransmitter, however, any substance of therapeutic significance is contemplated. In other embodiments disclosed herein, the device can comprise an exogenous marker gene. For purposes explained herein, a currently preferred selectable phenotype is one associated with a conditional defect in metabolic function such as, but not limited to, conditional auxotrophy.
In yet another currently preferred embodiment, the above-mentioned conditionally defective organism has a selectable phenotype due to the excision of a naturally-occurring genetic locus from its genome. In this embodiment, the organism is a transfectant, the genome of which comprises transfected DNA including a nucleotide sequence free of a marker gene and which is complementary to a wild-type nucleotide sequence flanking the locus in the wild-type organism, wherein the genetic locus is excised by homologous recombination with the transfected DNA. In a most currently preferred embodiment, the transfected DNA further comprises a nucleotide sequence defining a heterologous gene. Additionally, the present invention contemplates that a selectable phenotype can be generated by other genetic alterations such as, but not limited to, alterations achieved using transposon technologies. Typically, transposons cause loss of a genetic locus by interrupting the naturally-occurring nucleotide sequence, which can generate a selectable phenotype suitable for use with the present invention.
In a second aspect, the invention features a method of providing sustained delivery of an expression product to a host comprising the step of administering any one of the above-described sustained delivery devices to the host. In a currently preferred embodiment, the method of the invention further involves the step of controlling the detectable amount of device comprising the above-described organisms or expression product produced by the organisms. A currently preferred host is a mammal, although the present invention contemplates that any metazoan organism is a suitable host, including plants, insects and mammals.
In a third aspect, the invention features a method for producing any one of the above-described sustained delivery devices. The currently preferred method comprises the steps of excising a naturally-occurring genetic locus from the genome of a unicellular organism. This can be accomplished by transfecting the organism with DNA comprising nucleotide sequences complementary to wild-type sequences flanking the locus under conditions which promote excision of the locus, and then selecting for a conditionally defective phenotype generated by loss of the genetic locus. In a preferred embodiment particularly useful for delivery of a therapeutic substance, the transfecting step contemplates transfecting with DNA further comprising a heterologous gene. As described above, this heterologous gene can encode any therapeutically-significant substance such as, but not limited to, an enzyme, hormone or neurotransmitter. In another currently preferred embodiment, the transfecting step involves transfecting with DNA further comprising an exogenous marker gene. Yet another preferred method relies on transposon technologies to induce the loss of a genetic locus and the appearance of a selectable phenotype. It is understood that that any device prepared in accordance with these methods is within the scope of the present invention.
In short, the invention provides the art with a heretofore unappreciated method of producing unique devices for sustained delivery of therapeutically-significant products to a mammal. Moreover, in accordance with present teachings, these delivery devices can be controlled to provide effective dosages of immunostimulators and/or expression products with therapeutic/pharmaceutical benefits. Furthermore, these delivery devices can be targeted to specific tissues. Finally, as will be appreciated by the skilled artisan, the devices and methods of making and using the same disclosed herein can be used in human medical and veterinary applications, as well as insect and plant applications.
These and other objects, along with advantages and features of the invention disclosed herein, will be apparent from the description and claims that follow.
In its broadest aspects, the present invention provides the skilled artisan with the analytical tools and technical know-how sufficient to isolate conditionally defective organisms that can provide a delivery platform, preferably a sustained delivery platform, for therapeutically-significant genes and/or their expression products.
On the one hand, the skilled artisan will recognize that the delivery device of the instant invention can be exploited for its immunoprotective potential per se. In a related embodiment, the instant delivery device can be modified to enlarge or improve its inherent immunoprotective potential, such as by enabling the device to further express immunodominant or protective antigens, lymphokines or immunomodulatory substances. On the other hand, the present delivery device can also be used as a platform for administration of immunoprotective substances relevant to diseases induced by other, unrelated infectious agents or organisms. Still further, the skilled artisan will recognize that the present delivery device can be exploited as a general-purpose delivery platform by means of which any heterologous gene and/or its expression product can be provided to a host. Guidance provided herein accordingly will facilitate evaluation of diverse organisms as sustained delivery vehicles, thereby broadening the spectrum of potential therapeutic tools for amelioration and/or treatment of diseases.
In order to more clearly and concisely describe the subject matter of the claimed invention, the following definitions are intended to provide guidance as to the meaning of specific terms used in the following written description, examples and appended claims.
As used herein, the term xe2x80x9corganismxe2x80x9d includes any unicellular organism suitable for use in the devices and methods of the present invention. Haploid and diploid, including asexual diploid, unicellular organisms are contemplated. Particularly preferred unicellular organisms include protozoa, especially parasitic protozoa Set forth below is a non-limiting list of those unicellular protozoa contemplated to be within the scope of the present invention. Also set forth below is a discussion of generally preferred features and characteristics of the unicellular organisms most suitable for use in the present invention. As exemplified in Examples 2 and 8, respectively, two diploid genera currently preferred are Leishmania and Trypanosoma. Most currently preferred are the species L. major, L. tropica, L. aethipica, L. enrietti, L. panamaenisis, L. guyanensis, L. donovani, L. chagasi, L. infantum, T. cruzi, T. brucei and members of the trypanosomatid genus Endotrypanum such as E. monterogei and E. schaudinni. As also exemplified in Example 8, a currently preferred haploid genus is Toxoplasma; a most currently preferred species is T. gondii. Other currently preferred haploid genera include Plasmodium, Eimeria, and Cryptosporidia. A currently preferred genus of uncertain ploidy is Giardia. A most currently preferred species is G. lamblia. Other preferred genera of uncertain ploidy include Entamoeba, Acanthamoeba and Naegleria, and Microspoidia and Trichomona (T. vaginalis and T. foetus, in particular) generally.
The methods of the present invention permit production and isolation of a xe2x80x9cconditionally defectivexe2x80x9d unicellular organism. For purposes of the present invention, a conditionally defective organism must comprise a xe2x80x9cselectable phenotype,xe2x80x9d i.e., a phenotype generated by a genetic modification which permits identification and isolation of such an organism without resort to, or reliance on, markers encoded by exogenous genes. That is, a genetic modification has rendered it metabolically, nutritionally, reproductively, immunologically, pathogenically, and/or secretion disabled or dysfunctional relative to the wild-type organism. As contemplated herein, a conditionally defective organism is a genetically-modified unicellular organism whose tissue-specificity, growth and/or viability can be modulated by manipulating the conditions in which it is maintained, propagated and/or passaged. Consequently, one conditionally defective organism of the present invention can be dependent upon an exogenously provided substance or condition. Alternatively, in other embodiments, the organism is dependent upon the absence or lack of a particular substance or condition. As disclosed herein in Examples 2, 3, 5 and 7, this conditional disablement or defect can be exploited to the practitioner""s advantage. It will be understood that any genetic modification which gives rise to a selectable phenotype is within the scope of the present invention. Two currently preferred genetic modifications are described herein below, but are intended to be exemplary only.
In certain embodiments of the present invention, the above-described genetic modification is removal or excision of a genetic locus. The term xe2x80x9cgenetic locusxe2x80x9d is intended to include any nucleotide sequence, including a single nucleotide, within the genome of a unicellular organism. Preferably, a genetic locus corresponds to at least one allele of a gene. As exemplified below, a genetic locus can also preferably correspond to two alleles of a gene. The skilled artisan will appreciate the circumstances under which this distinction is operative. As already stated, removal or excision of a genetic locus is accomplished by transfecting the organism with DNA comprising sequences complementary to the naturally-occurring nucleotide sequences flanking either end of a genetic locus. The extent to which the transfecting sequences are complementary to the naturally-occurring sequences can vary. All that is required is that the transfecting sequences be complementary enough to permit a recombination event, for example homologous recombination, to occur. Similarly, the precise length of the flanking sequences can also vary. Again, all that is required is that the flanking sequences be of sufficient length to permit a recombination event to occur. Flanking sequences 400 bp long are effective, and sequences that are longer or shorter will also be effective. The skilled artisan will appreciate these fundamentals and can prepare suitable transfecting flanking sequences using only routine experimentation. Furthermore, only routine efforts are required to determine the primary nucleotide sequence of the DNA flanking either end of the genetic locus. Once the skilled artisan has selected the particular genetic locus which is to be removed or excised, the flanking sequences can be isolated by routine methods and their primary nucleotide sequence deduced by routine methods. For the purpose of this invention, it is understood that the extent of physical removal or excision of a nucleotide sequence comprising a particular genetic locus need only be enough to confer on the organism a selectable phenotype as defined herein.
Other embodiments of the present invention contemplate unicellular organisms rendered conditionally defective by interrupting, or otherwise disrupting, a naturally-occurring genetic locus. Methods of achieving such interruptions or disruptions using transposons are exemplified below in Example 4. Again, it is understood that the extent of, or precise manner of, physical interruption of a nucleotide sequence comprising a particular genetic locus need only be enough to confer on the organism a selectable phenotype. As used herein, the term xe2x80x9ctransposonxe2x80x9d refers to a discreet genetic element that insures its own maintenance by inserting into other autonomously maintained genetic elements. Transposons can be constructed to further encode substances of particular interest, for example a heterologous gene encoding an expression product of therapeutic interest. Transposons are useful tools for genetic manipulations such as deletions, inversions, and fusions, as is well known in the art. A transposon is a specific DNA segment with the ability to move as a unit in more or less random fashion from one genetic locus to another. Exemplary of specific transposons which may be used herein are transposons originally derived from insects, preferably members of the Tcl/mariner family, most preferably the mariner transposon element from Drosophila. As disclosed herein in Example 4, insertional inactivation mediated by a transposon can be a powerful way to generate mutants and gene fusions which would facilitate studies of gene function in an asexual diploid like Leishmania. The development of a mariner-based heterologous transposon system is a significant addition to the array of tools available for dissecting the genetic basis of relevant aspects of Leishmania biology, such as virulence and pathogenesis. The same system can be developed to ameliorate the restrictions of other asexual diploids using methods within the scope of the present invention.
As already explained, a xe2x80x9cselectable phenotypexe2x80x9d includes any phenotype generated by genetic modifications as described herein. That is, the present invention contemplates that a selectable phenotype arises upon loss of a naturally-occurring nucleotide sequence defining a genetic locus, preferably an allele, alternatively a gene. All that is required is that the resultant phenotype permits the skilled practitioner to identify and isolate the appropriately modified organism. A generalized discussion of selection paradigms and several examples of selectable phenotypes are set forth below. Currently preferred selectable phenotypes include conditional auxotrophy. An exemplary conditional auxotroph lacks all wild-type alleles of the DHFR-TS locus (for example, one allele in a haploid organism, and two alleles in a diploid organism). Other currently preferred selectable phenotypes include those associated with a conditional defect in metabolic function such as, but not limited to, nucleotide synthesis, metabolism or regulation. Still others are described hereinbelow.
As used herein, positive and negative selections are contemplated to select for and against the presence of a given genetic locus to the extent that the genetic locus is associated with a phenotype for which a positive or negative selection exists. A xe2x80x9cpositive selectionxe2x80x9d for a given phenotype is defined herein as a method of implementing a set of conditions wherein only cells that express the phenotype are isolated. A xe2x80x9cnegative selectionxe2x80x9d against a given phenotype is defined herein as a method of implementing a set of conditions wherein only cells which do not express the phenotype are isolated. A xe2x80x9cnegative selectionxe2x80x9d therefore selects against the presence, or for the absence, of the genetic locus associated with the phenotype. A genetic locus against which a negative selection can be applied is defined herein as a genetic locus which can act as a xe2x80x9cnegative marker.xe2x80x9d It will be understood that a xe2x80x9cpositive markerxe2x80x9d is defined analogously.
As exemplified herein in Example 2, the present invention permits isolation of a conditionally defective unicellular organism comprising a selectable phenotype without requiring reliance on exogenous marker genes. A xe2x80x9cmarker genexe2x80x9d includes any exogenous gene introduced via transfecting or transforming DNA sequences as disclosed herein, which is relied upon to identify and isolate a conditionally defective organism having a selectable phenotype as defined herein. As used herein, xe2x80x9cmarker genesxe2x80x9d such as exogenous antibiotic-resistance genes are genes generally considered by the art to be useful to monitor, identify and thereafter isolate genetically modified organisms. A xe2x80x9cmarker genexe2x80x9d falls within the broad definition of positive marker set forth above. Generally, such genes have heretofore been relied on in diploid organisms because no other selectable phenotype was appreciated or recognized. As demonstrated by the present invention, phenotypes can be generated and selected independently of exogenous marker genes. It is understood that the presence of a marker gene(s) per se does not affect exploitation of conditionally defective organisms as sustained delivery devices as exemplified below.
The term xe2x80x9csustained delivery devicexe2x80x9d means a device which comprises an isolated, conditionally defective organism, the genome of which lacks a naturally-occurring nucleotide sequence defining a genetic locus or a gene encoding a selectable phenotype. In certain preferred embodiments, the genome comprises a heterologous gene of interest. The genome can optionally further comprise an exogenous marker gene. It is understood that the sustained delivery device can be used at least to immunostimulate and/or deliver a therapeutically-significant substance to a host organism. In certain embodiments, a device can both immunostimulate and deliver a desired substance. Sustained delivery of an immunostimulating agent/or a therapeutic substance can be achieved using the devices of the present invention. The term xe2x80x9csustainedxe2x80x9d means of a period of time sufficient to at least achieve immunostimulation or expression of a desired substance in a host. In certain preferred embodiments, the present delivery device can persist in the host for whatever period of time is clinically required. As also. exemplified herein, sustained delivery can be accomplished by exploiting the organism""s conditional defect, such as by providing or depriving the host of a substance which can control the viability or biological competence of the organism. Exemplification of the foregoing can be found in Examples 5, 6, 7, and 8. Also contemplated is a type of sustained delivery which can be autoregulated by the organism. This is exemplified in Example 9.
In certain preferred embodiments, the sustained delivery device further comprises a xe2x80x9cgene of interest,xe2x80x9d most preferably a heterologous gene encoding an expression product of interest. xe2x80x9cHeterologousxe2x80x9d means not naturally-occurring in the genome of the organism, including gene copy number. Numerous such genes and their expression products within the scope of this invention are listed below. As used herein, xe2x80x9cexpression productxe2x80x9d means any product expressed by the organism, the sustained delivery of which is desired. An expression product can be encoded by a homologous gene, i.e., a gene which is naturally-occurring in the organism, or an expression product can be encoded by a heterologous gene as already explained. An example of a desirable expression product encoded by a homologous gene is an expression product which makes the organism per se a suitable immunostimulating agent or vaccine. Thus, it is understood that the term expression product is not limited in its use herein. When required, expression products can be detected using routine means available in the art. Expression products contemplated herein include, but are not limited to: DNAs, RNAs, oligonucleotides, proteins, peptides, carbohydrates, lipids, nucleosides, amino acids, steroids, fatty acids, vitamins and antibiotics including anti-viral substances. It is understood that heterologous genes can be introduced into the genome concomitantly with, or independently of, the transfecting or transforming sequences described above. Additionally, a heterologous gene can reside on a plasmid and be introduced thereby.
As used herein, the term xe2x80x9cimmunostimulatexe2x80x9d means to stimulate the immune system of the recipient. Immunostimulation results in one or more functions within the immune system being induced or increased and, in certain embodiments, directed towards an immunostimulating agent. Immunostimulating agents include agents such as conventional vaccines, or the vaccine-type devices disclosed herein in Examples 5 and 6. By way of example only, immunostimulation can be measured by the production of antibodies. As used herein, xe2x80x9cvaccinexe2x80x9d is understood to mean any substance, device or agent used to stimulate the immune system of a living organism so that some protection against future disease and/or harm is provided, even if transiently. Immunization refers to the process of inducing antibodies and/or cellular immune responses. It is understood that vaccine-type devices of the nature disclosed and exemplified herein are likely to produce a broad range of immune responses in addition to immunoglobulin production, for example, cellular and humoral immunity.
In their broadest applications, the xe2x80x9cnull-targetingxe2x80x9d methods of the invention can be used to generate homozygous deletions of any genetic locus against which negative selection can be applied. Null-targeting refers to the process of removing a genetic locus, for example by homologous gene replacement, using appropriate constructs lacking the genetic locus, such that no DNA necessarily replaces the removed locus. A preferred aspect of the invention is that homozygous deletions can be generated without the concomitant introduction of any exogenous marker gene, into the chromosomal site of the deletion. A negative selection can be applied against a genetic locus if the genetic locus is responsible for (or encodes) a phenotype against which a negative selection exists or can be implemented. Using methods of negative selection against a given phenotype comprises implementing a set of conditions under which cells can be isolated and subsequently grown as clonal populations of cell which do not express the phenotype. As a general example, a negative selection against the phenotype of expressing a given gene product comprises exposing cells to conditions that allow the isolation of cells which do not express the gene product. The isolated cells can subsequently be grown as clonal populations of cells which do not express the gene product.
The following non-limiting general examples of phenotypes and negative selections will guide the artisan of ordinary skill in the practice of methods according to the invention. It is understood that in the following examples, the phenotypes are non-essential phenotypes; that is, the absence of the phenotype is not lethal to the cells under the conditions of the negative selection. Expression of a given metabolic enzyme can be selected against by growing cells in the presence of a compound that, when metabolized by the enzyme, becomes toxic and kills the cells. Under these conditions, only cells which do not express the enzyme, and therefore do not metabolize the compound, will grow. Expression of a given metabolic enzyme can also be selected against by growing cells in the presence of a compound that, when metabolized by the enzyme, imparts an observable phenotype to the cells, for example a specific color or fluorescence. Under these conditions, only cells which do not express the enzyme are non-colored or non-fluorescent. These cells can be identified and isolated by methods well known in the art, such as growing under conditions allowing the formation of isolated colonies, or by fluorescence activated cell sorting. In a related example, selection against a given metabolic enzyme can be accomplished by growing cells under conditions which alter the function of the enzyme in such a way that deleterious or lethal metabolites accumulate in the cells. Under these conditions, only cells that do not express the enzyme will grow well, and these cells can thereby be isolated using methods known in the art. These examples therefore allow cells which do not express the enzyme to be isolated from cells which do express the enzyme. A further general example of a negative selection comprises identifying cells which do not express a given antigenic product. This can be accomplished using colony blotting and Western blotting procedures well known in the art. Yet another general example comprises cell-surface markers and marker-specific antibodies conjugated with toxins.
The following non-limiting examples of genes can be used as negative markers according to methods of the invention: adenine (APRT), hypoxanthine/guanine (HGPRT), hypoxanthine/guanine/xanthine (HXGPRT), uracil phosphoribosyl transferase (UPRT), dihydrofolate reductase-thymidine synthetase (DHFR-TS), thymidine kinase (TK) and orotodine 5xe2x80x2-phosphate- decarboxylase. These markers can be selected against using methods known in the art which illustrate some of the general principles of negative selection described above. A specific example of using DHFR-TS as a negative marker for null-targeting in Leishmania is provided in Example 2.
The null-targeting methods of the invention are not limited to phenotypes against which a negative selection can be applied. According to methods of the invention, it is possible to generate null-targeted deletions of any locus on the chromosome, provided that the deletion is not lethal to the cell. The method comprises the step of first introducing a known negative marker into the locus at which a null-targeted deletion is desired. This is achieved by transfecting the cell with a linear piece of DNA comprising a negative marker, a positive marker and flanking sequences which are complementary to sequences flanking the genetic locus to be deleted. Positive selection for the positive marker is used to isolate cells in which the transfected DNA has undergone homologous recombination with the chromosomal DNA, resulting in the targeted locus being replaced by the positive and negative markers. In a second step, the null-targeted deletion is generated according to methods described herein. Specifically, the isolated cell line, containing both the positive and negative markers, is transfected with DNA comprising the above-mentioned flanking sequences, with no other sequence between the flanking sequences being required. Negative selection against the negative marker is used to isolate null-targeted deletion cells in which the transfected DNA has undergone homologous recombination with the chromosomal DNA, resulting in the positive and negative markers being excised. The positive marker used in this method can be any gene for which a positive selection can be applied, for example any of the drug resistance markers well known in the art. A marker may be used which can act both as a positive marker and as a negative marker. A specific example of the use of this type of marker is provided in Example 3. Alternatively, the positive and negative markers may be different. The positive and negative markers may be derived from the species being null-targeted. Alternatively, one or both of the markers may be derived from a different species of organism. For the marker to be most useful, the organism being null-targeted preferably does not express the phenotype associated with the marker. If, for example, the marker is a gene derived from the species being null-targeted, then the cells used according to this method are preferably deficient in this marker gene, due to a mutation, preferably a deletion in this marker gene. It will be appreciated by the skilled artisan that this method alone or in combination with other methods disclosed herein, and known in the art, can be used to null-target a haploid organism, null target a single allele of a diploid organism, null target both alleles of a diploid organism or null-target any number of alleles of an organism of higher ploidy. Non-limiting examples are provided in Example 8.
Accordingly, the present invention provides the technical know-how to construct and isolate conditionally defective organisms. Such organisms can then be exploited to deliver numerous and functionally varied therapeutic substances. By way of example, the following categories of biotechnology and/or therapeutic substances are contemplated. These categories include: metabolic enzymes, antisense molecules, clotting factors, colony stimulating factors, erythropoietins, growth factors, human growth hormones, interferons, interleukins, monoclonal antibodies, recombinant soluble receptors, tissue plasminogen activators, to name but a few.
More specifically, examples of antisense therapies currently under development suitable for use with the present invention include: HIV antisense, cancer antisense and inflammatory disease antisense. Clotting factors can include recombinant human factors currently under development for treatment of hemophilia A and B. Exemplary colony stimulating factors include: GM-CSF, and rG-CSF currently considered useful for treatment of infectious diseases, hemophiliac disorders, and HIV infection. Among the growth factors, those contemplated herein include the art-recognized compounds known as: transforming growth factor-beta, brain-derived neurotrophic factor, transferrin, insulin-like growth factor, nerve growth factor, neurotrophin-3, recombinant human platelet-derived growth factor-BB, and recombinant insulin-like growth factor-I/binding protein-3 to name but a few. Human growth hormones include human growth hormone releasing peptide, somatropin for injection, and recombinant variants thereof. The category of interferons include interferon gamma-1b, interferon alfa-n3, gamma interferon, consensus interferon, recombinant interferon beta, to name but a few of the more well described species of interferons. With respect to the interleukins, substances such as liposomal IL-2, recombinant human interleukin-1, recombinant interleukin-2, recombinant human interleukin-3, glycosylated recombinant human interleukin-6, and IL-12 and its natural and synthetic variants, are also contemplated as suitable for use with the present invention.
Currently available and well described monoclonal antibodies useful with the present. invention include: monoclonal antibodies directed to breast cancer, HIV, liver and germ cell cancers, allergic diseases, asthma, metastatic or recurrent colorectal cancer, rheumatoid arthritis, ovarian cancer, gram-negative sepsis, multiple sclerosis, solid tumors, metastatic cancer, leukemia and metastatic melanoma, acute myeloid leukemia, respiratory syncytial virus disease, B-cell lymphomas, B-cell leukemias, T-cell malignancies, small-cell lung cancer, renal diseases, prostate adenocarcinoma, acute CMV disease, to name but a few. Recombinant soluble receptors, for indications such as asthma, treatment of rhinovirus induced common cold, septic shock, severe sepsis, rheumatoid arthritis, multiple sclerosis, respiratory distress syndrome, are contemplated herein.
Other contemplated substances include: recombinant human glucagon, recombinant human follicle-stimulating hormone, insulin, human corticotropin-releasing hormone, insulinotropin, recombinant human leutinizing hormone, recombinant human parathyroid hormone, stem cell factor, recombinant human thyroid stimulating hormone, tissue factor pathway inhibitor, to name but a few. As stated earlier, these particular substances merely illustrate the broad categories of biotechnology and/or therapeutic indications with which the present invention can be practiced, and the invention is not limited to practice therewith. A currently preferred substance is insulin as exemplified in Example 9.
Generally speaking, the devices and methods of this invention can be used to deliver, preferably in a sustained manner, a diverse variety of substances. All categories of ligands, peptides, proteins, lipids, fatty acids, steroids, catacholamines, neurotransmitters, immunostimulants, carbohydrates, amino acids, neurostimulatory factors, human growth factors, and cytokines are contemplated. Any substance deemed to have therapeutic value is contemplated herein. Currently, substances such as insulin, gamma interferon, BMPs, tissue plasminogen activator, beta interferon, Ceredase(copyright), Cerezyme(copyright), erythropoietin, GM-CSF, G-CSF, DNase, and Factor VIII are among the preferred art-recognized substances of current therapeutic interests. Other currently preferred substances would include those suitable for treating the following selected diseases such as, but not limited to, osteoporosis, diabetes, cancer, severe anemia, short stature, and hemophilia Gaucher""s disease as treated by Ceredase(copyright) (and/or its naturally-occurring and synthetic variants) and diabetes as treated with insulin (and/or its naturally-occurring and synthetic variants) are two currently-preferred disease-therapy paradigms.
It is contemplated that, in view of the human genome initiative, very many more genes of therapeutic value will be sequenced. Consequently, the identification of many proteins with therapeutic value will be forthcoming and are contemplated to be within the scope of the present invention. Particularly preferred substances include, but are not limited to, those biotechnology drugs currently approved for use in humans. Such drugs include: gamma interferon, interferon alpha-N3, recombinant interferon beta I-A, recombinant interferon beta 1-B, recombinant alglucerase, recombinant imiglucerase, CMV immunglobulin, daunorubidin, doxorubicin, somatrophin, recombinant human insulin, alpha-interferon, yeast derived GM-CSF, pegaspargase, human growth hormones, DNase, recombinant antihemophilic factor, recombinant hepatitis B, to name but a few.
As exemplified herein, the present invention also provides a method for delivering a gene of interest to a mammal. Numerous heterologous genes have been identified in the art and their delivery to a patient can be accomplished using the methods and devices of the present invention. For example, gene delivery in accordance with the present invention can be used to treat the following indications: cystic fibrosis, sinusitis, HIV, colon cancer, metastatic renal cell carcinoma, disseminated malignant melanoma, neuroblastoma, Gaucher""s disease, breast cancer, melanoma, non-small cell lung cancer, and clinical equivalents thereof. A currently preferred disease is Gaucher""s disease as exemplified in Example 9.
As exemplified herein in Example 5, the present invention also provides a device for delivering an immunostimulant to a mammal. In certain embodiments, this immunostimulating property of the instant device serves to vaccinate the recipient. Accordingly, the present invention can serve as an improvement for a conventional vaccine for the following diseases and/or conditions: pediatric pertussis, HIV infection, multiple sclerosis, melanoma, breast cancer, diarrheal diseases and gastritis, CMV virus, prostate and ovarian cancers, diphtheria, tetanus, cancers such as colorectal, stomach, and pancreatic, peptic ulcers, melanoma, hepatitis B, herpes simplex, influenza, psoriasis, lyme disease, infectious diseases generally, respiratory syncytial virus, and various protozoa and parasitic protozoa such as Leishmania, to name but a few.
Additionally, it will be appreciated by the skilled artisan that this invention also provides methods and devices for desensitizing an organism to an allergen. By way of example, sustained delivery devices can be constructed to express or produce an allergen. As used herein, allergens are substances that cause allergic reactions. It will be appreciated that many different materials are allergens such as, but not limited to, animal dander and pollen. It is possible to induce tolerance to an allergen in an organism that normally shows an allergic response. Methods of inducing tolerance are well known and generally comprise administering allergen to the organism in increasing dosages. Thus, in one further embodiment, when the immunostimulating component of the delivery device is an allergen of the host, the present invention permits the skilled practitioner to develop an exposure regimen designed to specifically desensitize the allergic host. It is contemplated that embodiments of this nature can also be used to desensitize an auto-allergic response in an organism afflicted with auto-immune disease.
As already stated, the preferred organism of the present invention is a unicellular organism. In certain embodiments, a diploid organism is preferred; in others, a haploid organism. Currently, one of the preferred unicellular organisms is a protozoa. According to M. A. Sleigh (1991 Parasitic Protozoa, 2nd edition, volume 1, pages 1-53, eds. J. P. Kreier and J. R. Baker; Academic Press, Inc., New York), protozoa can be categorized by certain distinguishing structural characteristics. The four phylum are: Sarcomastigophora which includes organisms having pseudopodia or flagella as locomotor organelles, including the amoebae and flagellate protozoan parasites. Ciliophora includes organisms bearing cilia; Apicomplexa includes a large and diverse group of organisms including the intestinal, blood, and tissue dwelling coccidians; and finally, Microspora includes spore forming organisms, among which are human parasites.
Among the flagellate protozoa, there is a subgroup called kinetoplastids (trypanosomes and their allies). Most of the 600 or so species of kinetoplastids are parasites, including important genera such as Trypanosoma and Leishmania which parasitize vertebrates, including humans. These genera are among two of the currently preferred genera of diploid organisms suitable for practice with the instant invention. Other genera can be found as gut parasites in insects and as parasites in plants. Accordingly, members of such genera can be used to practice the instant invention when the desired host is an insect or a plant. Among those spore forming groups of protozoa, there are three subcategories. They include microspora, sporozoa, and mixospora. The microspora are intracellular parasites. There are a number of microspora genera which parasitize invertebrates such as Encephalitozoon in mammals, including humane with immune deficiency, and Glugea in fish. Another wide spread genera is Nosena, the species of which cause important diseases in insects of economic importance such as bees and silk worms. Again, in applications involving hosts such as non-human vertebrates or insects, the delivery device of the present invention can be exploited most successfully. Among the sporozoa category, there are numerous intestinal, blood, and tissue dwelling coccidians suitable for use with the present invention. Particularly useful coccidians include Cryptosporidium, Isospora, Toxoplasma, Sarcocystis, and Plasmodium. Certain species of genera of Eimeria and Isospora are important pathogens of domestic animals. Toxoplasma is a widely distributed parasite of mammals that is now regarded by certain artisans as a member of the genes Isospora. Toxoplasma is a currently preferred haploid genera suitable for use with the present invention. Finally, members of the subcategory mixospora include protozoa commonly considered parasitic of fish. For example, it is currently believed that a member of the mixospora category is responsible for xe2x80x9cwhirling diseasexe2x80x9d in trout. The skilled artisan will appreciate that the instant invention would be applicable to control such a disease.
While a generally useful feature of currently preferred organisms is that they can be cultured in vitro, this is not a requirement. Among the above-referenced diploid genera, two currently preferred are Leishmania and Trypanosoma, both blood and tissue flagellates reside in humans. Both genera can, in certain stages of their life cycle, be propagated in culture. Moreover, numerous aspects of the molecular biology of protein processing and expression have been studied in these genera. Features shared with higher eucaryotes include synthesis of capped polyadenylated cytoplasmic RNA; a typical ribosome and protein synthetic apparatus; and, a general protein secretory apparatus including endoplasmic reticulum, golgi apparatus and exo- and endo-cytosis.
In the genera Leishmania, several species can cause visceral disease and reside intracellularly, for example, in lymph nodes, liver, spleen, bone marrow, etc. Other species of Leishmania cause cutaneous and mucocutaneous diseases. Such species are found intracellularly and extracellularly in skin and mucous membranes of humans. Within the genera Trypanosoma, it is well known that certain species reside intracellularly in viscera, mycocardium, and brain in humans. During certain stages of their developmental cycle, these species may also reside in blood, lymph nodes, cerebro-spinal fluid, depending upon whether the organism is residing in the host or the vector.
A particularly preferred haploid genera is Toxoplasma. Toxoplasma is an obligate intracellular parasite. It is well known that Toxoplasma is culturable. All of the known protein-coding genes are present in single-copy. Gene expression in Toxoplasma is apparently conventional; that is, promoters are defined and thematically similar to higher eucaryotes. (See, for example, 1995 Molecular Approaches to Parasitology, pp. 211-225, Boothroyd et al. (eds. J. C. Boothroyd and R. Komuniecki; J. Wiley and Sons, N.Y.).) An especially preferred species that is well-characterized is T. gondii. Toxoplasma is distributed world-wide and resides in various cells, tissues and fluids of the host. During certain stages, the organism can be found in the central nervous system, skeletal and cardiac muscles, and visceral organs and tissues. Domestic cats are important reservoirs for human infection of this organism. For example, human infection can be transmitted in a variety of ways: handling infected cat feces; ingestion of meat from infected animals such as pork and lamb; transplacental transmission; transfusion with infected blood; and, via organ transplantation from infected donors. Of particular interest herein is the fact that toxoplasmic encephalitis is the most common opportunistic parasitic infection of the central nervous system in patients with AIDS.
As already stated, two particularly preferred genera of protozoa include Leishmania and Toxoplasma. Another currently preferred genera is Giardia. In certain embodiments, other pathogenic amoebae are contemplated. Specifically, the currently preferred unicellular diploid organisms include, but are not limited to: Protozoans of the family Trypanosomatidae (Trypanosomoa cruzi, T. brucei, Leishmania spp (including subgenus Leishmania and Viannia; examples include L. major, L. tropica, L. aeithiopica, L. entrietti, L. mexicana, L. amazonesis, L. donovani, L. chagasi, L. infantum, L. braziliensis, L. panamaensis, L. guyanensis, and others). The currently preferred haploid organisms include, but are not limited to: members of Apicomplexa (Toxoplasma gondii, Plasmodium, Eimeria, Cryptosporidia, and others). Protozoans of uncertain ploidy include, but are not limited to: amoebae including Entamoeba spp, Acanthamoeba spp, Naegleria spp, Giardia lamblia, and members of the phyla including Microspordia and the trichomonads (Trichomonas vaginalis, Tritrichomonas foetus).
Other suitable protozoans known to have human hosts include: Entamoeba histolytica, Entamoeba hartmanni, Entamoeba coli, Entamoeba polecki, Endolimax nana, Iodamoeba buetschlii, Naegleria fowleri, Acanthamoeba species, Dientamoeba fragilis, Giardia lamblia, Chilomastix mesnili, Trichomonas vaginalis, Pentatrichomonas hominis, Enteromonas hominis, Balantidium coli, Blastocystis hominis, Isospora belli, Sarcocystis species, Cryptosporidium parvum, Enterocytozoon bieneusi, Toxoplasma gondii, Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium species, Babesia microti, B. equi, B. bigemina, Trypanosoma b. gambiense, T. b. rhodesiense, T. cruzi, T. rangeli, Leishmania species and Pneumocystis carinii. 
In view of the present disclosure, the skilled artisan will appreciate that the sustained delivery device exemplified herein is particularly suitable for tissue-targeted applications. Each of the above-described organisms has a tissue preference and each can be exploited by judicious practice and/or routine adaption of the present invention. Tissue-specificity is further discussed in Example 9.
One particularly preferred feature and advantage of the organisms contemplated herein is an ability to escape from the host immune response. The particular mechanism by which this immune evasion is accomplished is not relevant or critical to practice of the present invention. All that is required is that the particular organism be successful, at least in part, in its efforts to invade and reside in the host. The skilled artisan will appreciate that persistence in the host can be, in part, controlled by selection of the particular organism administered to the host. That is, the organisms contemplated herein collectively demonstrate a broad spectrum of persistence in the host. Practice of the present invention permits discrete modification of a particular organism which already has an ability to live and persist in, even if for a limited time, the host body such that it can deliver therapeutic genes/proteins. The above-described organisms have developed this preferred ability to inhabit their host""s body. In certain preferred embodiments, the method of making the delivery device described herein can permit controlling or modifying the virulent properties of a particular organism which, in part, permit it to invade its preferred host. In so doing, any of the above-described pathogenic-type organisms can be rendered partially or wholly avirulent as well as conditionally defective. In essence, practice of the present invention results in production and exploitation of xe2x80x9cdomesticatedxe2x80x9d protozoa or protozoa parasites.
Evidence that the above-described organisms can be genetically modified using routine methods can be derived from the numerous publications describing protocols, reagents, and other experimental parameters useful therefor. (For example, see 1993 Protocols in Molecular Parasitology, ed. J. E. Hyde; Humana Press, Inc., New Jersey; and 1995 Molecular Approaches to Parasitology, eds. J. C. Boothroyd and R. Komuniecki; John Wiley and Sons, Inc., New York, the disclosures of both of which are herein incorporated by reference.) Some of the genera routinely studied using art-recognized genetic and molecular techniques include, but are not confined to, Trypanasoma, Leishmania,. Plasmodium, Schistosoma, Giardia, Theileria, and Toxoplasma. In view of the widely publicized materials and methods for genetically modifying organisms such as those described above, it will be appreciated that merely routine experimentation and routine skill is required to practice this present invention with a particular haploid/diploid unicellular organism, including protozoan and/or parasitic organisms.
It is understood that the present invention can be used for applications in which the host is a metazoan organism. That is, a multi-cellular organism including plants and insects as well as mammals is a suitable host. In such applications, the particular organism which serves as the sustained delivery device is one capable of establishing itself in such a host. Examples of such organisms have been described elsewhere herein. Accordingly, the methods of sustained delivery of a conditionally defective organism described herein are not limited to methods of delivering such an organism to a mammal. A mammal is simply an example of a multi-cellular, metazoan organism with which the present invention can be practiced. Thus, in its broadest respects, the present invention features devices and methods of making and using the same for sustained delivery of a therapeutically significant gene/expression product to a metazoan organism, preferably mammals, plants, and insects, and most preferably mammals. Currently, two particularly preferred mammals include humans and primates.
Additionally, the present invention contemplates that the recipient of the disclosed device includes all vertebrates, for example, mammals, including domestic animals and humans, various species of birds, including domestic birds, particularly those of commercial importance. In addition, mollusks and certain other invertebrates have a primitive immune system and are included as a recipient. As used herein, a vertebrate is any member of the subphylum Vertebrata that includes fishes, amphibians, reptiles, birds, and mammals, all of which are characterized by a segmented bone and cartilaginous spinal column. All vertebrates have a functional immune system and respond physiologically by evidencing immunostimulation. An invertebrate is any member of the animal kingdom excluding the vertebrates. Such animals have no back bone or spinal column. Classification includes all animals except fishes, amphibians, reptiles, birds and mammals. Exemplary of such invertebrates are shellfish and mollusks and other related animals. Although the use of immunostimulating agents and delivery of heterologous genes/expression products to invertebrate animals has heretofore not been well documented, one skilled in the art will recognize the applicability of the subject invention to said invertebrates.
Thus, the present invention also provides a method for exploiting protozoa as delivery vehicles for use in insects. It is anticipated that the methods and devices of the present invention will involve preparing conditionally defective diploid organisms, such as protozoa ordinarily stably associated with a particular insect species. In this regard, the present methods and devices can be used to introduce genes corresponding to antibiotics, for example, which have highly selective anti-insecticidal activity. Still another preferred embodiment provides a device for use in plants. For example, it is anticipated that the methods and devices of the present invention can be used to introduce anti-herbicidal and anti-insecticidal activities.